The precise role of phosphorylated heat shock protein (HSP) 27 (p-HSP27) in pancreatic cancer remains to be elucidated. The aim of this study was to investigate whether the expression of p-HSP27 predicts the prognosis of patients with pancreatic cancer.We retrospectively assessed 49 biopsied pancreatic cancer tissue samples that were obtained prior to the treatment with gemcitabine. The correlations between p-HSP27 and the clinicopathological characteristics were analyzed.n = 18) than those with low p-HSP27  (P = 0.0158). A multivariate Cox proportional hazards regression analysis revealed that low p-HSP27 predicted a worse prognosis.p-HSP27 was not correlated with the response to chemotherapy or histological type. However, the median survival time was significantly longer in the patients with high p-HSP27 (275 days, Higher p-HSP27 expression before chemotherapy was correlated with better survival, indicating that p-HSP27 expression could be used to predict the prognosis of pancreatic cancer. Pancreatic cancer, which causes approximately 266,000 deaths per year, is the eighth leading cause of cancer-related deaths worldwide . SurgicaHeat shock proteins (HSPs) were first discovered as a family of proteins that are induced by heat shock, and other chemical and physical stresses . HSPs acHSP27 undergoes various types of post-translational modifications such as phosphorylation, in which the cellular functions of HSP27 are modulated . Human HOn the other hand, we previously reported that p-HSP27, which is induced by gemcitabine via p38MAPK-MAPKAPK-2, plays a key role in the suppression of pancreatic cancer cell growth in stably transfected-mutant HSP27 pancreatic cancer cell lines . In the We retrospectively analyzed the HSP27 expression level score and positive p-HSP27 ratios in biopsy specimens obtained by EUS-FNA. While the positive expression of HSP27 was found in all of the 49 patients 100%), the intensity of the HSP27 staining differed considerably among the cases . However, when the cut-off value was set at 76% for a positive p-HSP27 ratio, there was a significant difference .We investigated whether the baseline characteristics of the enrolled patients and their HSP27 expression level score or positive p-HSP27 ratio were associated with survival in a univariate analysis using a log-rank test. The patients were divided into two groups according to the median age. When the patients were divided into two groups according to their survival period (more than 1 year or less than one year) in an ROC analysis, the area under the curve (AUC) values for the HSP27 expression level score and a positive p-HSP27 ratio were 0.56 and 0.67, respectively , metastasis , histological type (poor) , and the response to chemotherapy (PD)  also remained to be significant predictors for overall survival  and the histological type of poorly differentiated adenocarcinoma  were found to be independently correlated with worse survival , and the response to chemotherapy (PD) were significant (data not shown). A multivariate Cox proportional hazards regression analysis was therefore performed for these factors. As a result, a low positive p-HSP27 ratio  = 1.089–4.523; The results of the present study showed the first evidence that a low positive p-HSP27 ratio, which was observed prior to treatment with gemcitabine-based chemotherapy, was significantly correlated with the presence of metastasis Figure  and poorA question arises here concerning the biological meaning and the mechanism underlying the superior survival of pancreatic cancer patients with increased levels of p-HSP27. Because p-HSP27 is associated with the induction of apoptosis in pancreatic cancer cells , the incIn the present study, the status of HSP27 phosphorylation before chemotherapy was not associated with the response to gemcitabine, although a previous study suggested that p-HSP27 might contribute to gemcitabine resistance . In addiIn addition, it might also be important to determine whether the expression levels of HSP27 and p-HSP27 were altered after gemcitabine-based chemotherapy. There is a possibility that gemcitabine sensitivity and the induction of apoptosis are increased at the pancreatic tissue level when a high positive p-HSP27 ratio is present . It is mIn conclusion, a positive p-HSP27 ratio was significantly associated with a longer survival time. In the near future, p-HSP27 could therefore be used as a prognostic marker for patients with pancreatic cancer.n = 22; male, n = 27; median age, 68 years [range: 31–86 years]) who underwent EUS-FNA at the department of Gastroenterology, Gifu University Hospital between September 2004 and October 2011. The patients' demographic and clinicopathological characteristics are shown in Table The study population included 49 pancreatic cancer patients . The echoendoscope was connected to a processor with a color Doppler function . FNA was performed with a 19-, 22- or 25-gauge needle  under direct EUS guidance.2O2 in methanol for 10 min to inhibit endogenous peroxidase activity. Sections were then immersed in 50 mM citrate buffer (pH 6.0), and heated in a microwave oven for 1 min. Next, each section was sequentially treated with biotinylated secondary antibodies and streptavidin-peroxidase complex . Finally, the immune complexes were visualized with 3, 3′-diaminobenzidine tetrahydrochloride as a chromogen. Mayer's hematoxylin was used as a counterstaining agent.The immunohistochemical staining of the biopsied tissues was performed with the streptavidin-biotin complex method to investigate the levels of HSP27 and p-HSP27, and their localization. The primary antibodies were anti-HSP27 and anti-p-HSP27 (Ser-82) antibodies, respectively . Briefly, deparaffinized sections were first treated with 3% HHSP27 expression level score: We calculated the HSP27 expression level score using the quantitative-histogram scoring method . We grad® 8.0 software program . The values are expressed as the number of patients or the median (range). The median values were compared using Student's t-test. A receiver operating characteristic (ROC) analysis was performed to determine the cutoff values for the HSP27 expression level score, the positive p-HSP27 ratio. Survival rates were calculated using the Kaplan-Meier method and the difference between the curves was analyzed using the log-rank test. A multivariate Cox proportional hazards regression analysis was performed to test prognostic significance. Forward stepwise selection was used to obtain an efficient predictive model. All P values were reported. P values of < 0.05 were considered to be statistically significant.All analyses were conducted using the JMP


When the NPB thickness is reduced from 2to 0.3 μm and with a thin 10-nm CGL, the hole transientsignal still shows non-dispersive properties under various applied fields, and thusthe hole mobility is determined accordingly. Only 1-μm NPB is requiredfor determining the electron mobility by using the proposed CGL. Both thethicknesses are the thinnest value reported to data. In addition, the flexibility offabrication process of small molecules can deposit the proposed CGL underneath andatop the material under test. Therefore, this technique is applicable tosmall-molecule and polymeric materials. We also propose a new approach to design theTOF sample using an optical simulation. These results strongly demonstrate that theproposed technique is valuable tool in determining the carrier mobility and may spuradditional research in this field.Time-of-flight (TOF) measurements typically require a sample thickness of severalmicrometers for determining the carrier mobility, thus rendering the applicabilityinefficient and unreliable because the sample thicknesses are orders of magnitudehigher than those in real optoelectronic devices. Here, we use subphthalocyanine(SubPc):C Organic materials enabled fabricating optoelectronic devices onto large-area and flexiblesubstrates in low-cost approaches12346789101112131416171819202223242526272829303132The carrier mobility can be characterized using time-of-flight (TOF) measurement,space-charge limited current (SCLC) model, transient electroluminescence (EL)measurement, and field-effect transistors (FETs)343536384143474849374751et al. used silicon as substrates for measuring the carrier mobility of ELmaterials, thus showing that the wide wavelength selection of silicon to Nd:YAG lasercan act as an efficient CGL and reduces the required thickness of organic layers to thetypical value that used in organic electronic devices5137596164656667Despite these deficiencies, it is still favorable to use TOF measurement in observing theeffects of the molecular structures, trap states, scattering centers, and dispersivityon the carrier transport475354555657243752535859606170 acceptor69et al. demonstrated an improved device fabrication process toenhance the device efficiency70 for the use in CGLs. In the current study, wedetermined the hole mobility of 0.3-μmN,N′-diphenyl-N,N′-bis-4,4′-diamine(NPB) by using the proposed CGL, which provides a well-confined charge-generation width,thus leading to clear non-dispersive properties under various applied voltages. Althougha 1-μm NPB was required for obtaining the non-dispersive transient signal,the electron mobility was determined and in good agreement with previous studies. Inaddition, we proposed a new concept for designing the TOF sample through simplysimulating the power-dissipation profiles in the samples. The proposed CGL wasthermal-evaporated and can be deposited underneath or atop the mater under test.Therefore, the proposed technique was applicable to determine the carrier mobility ofpolymeric materials.As an efficient CGL, high absorption and carrier transport are prerequisite for anincreased number of photo-excited carriers that contributes to the transient signalthrough the material under test. These features are present in the photosensitizedorganic materials that used in OPV devices. In our previous studies, we have fabricateda series of devices with high performance on the basis of the subphthalocyanine (SubPc)donor and C3 (15 nm)/SubPc(5 nm)/SubPc:C70 /C70(43-x nm)/bathocuproine (BCP) (8 nm)/Al, where xis 10, 15, 20, 25, and 30 nm, for studying the effects of theSubPc:C70 thickness on the charge generation and transportproperties. The result may be misinterpreted if a single layer of C70was not used because the first optical-field maximum is unlikely to occur withinthe active layer, as predicted by the optical modeling (J-V)characteristics under AM 1.5G illumination at100 mW cm−2 for thedevices with various structures. The error bar representing the standarddeviation is provided at each data point. The error of fill factor (FF) waslower when the SubPc:C70 thickness increased, probably because of theinhomogeneity of the thin film formation at a thinner thickness. Nevertheless,the thickness variation has a minor influence on FF. The difference inopen-circuit voltage (VOC) is probably due to the logarithmicrelation between VOC and short-circuit current density(JSC) and the carrier recombination caused by anincomplete interpenetrating network between the SubPc and the C70.Such issue, however, is beyond the scope of the current study. The mainparameter of special interest here is JSC, which represents aphoton absorption and charge transport properties The parameterJSC showed a considerable variation when variousSubPc:C70 thickness were used. 70. To determine the variation in JSC,external quantum efficiency (EQE) spectra of the OPV devices were measured, asshown in JSC is theintegral of the product of the EQE and AM 1.5G solar spectrum, the averaged EQEin a wavelength range of 500 to 600 nm was directly proportional toJSC. Although the EQE decreased with theSubPc:C70 thickness, these values seem sufficient to allow thecarriers for flowing through the material under test in the TOF measurement.These results indicate that the mixture of the SubPc and C70 is apromising candidate for the use in CGLs, and its thickness allows room formodulating according to the tested material thickness.In the TOF measurement, a thickness-tunable property is preferable withoutcompensation on both the absorption and transport for controlling thecharge-generation width because the thickness of a CGL must be considerablythinner than that of the material under test. Therefore, in the current study,OPV devices were fabricated based on a structure of indium-tin-oxide(ITO)/MoOmodeling . Figure2 laser) to induce the photogenerated carriers for measuring thecarrier transient signal of the NPB without using a CGL6360737475767778796073757670 would be apromising CGL upon the incident wavelength of 532 nm, as thephoto-response and charge-generation ability of the OPV device composed of theSubPc and C70 was extraordinarily prominent according to the highabsorption, EQE, and internal quantum efficiency (IQE)  levelsat the SubPc:C70/NPB interface. However, the lowest unoccupiedmolecular orbital (LUMO) levels between the CGL and the NPB were not matched,thus resulting in large injection barrier that may impede the electron injectionacross the SubPc:C70/NPB interface. This issue may be resolvedthrough inserting an injection layer and/or applying a relatively high bias topromote the electron injection from the CGL into the NPB, the latter one hasbeen proven to be feasible and thereby the electron mobility was determined asshown in the previous studyL under an applied voltage (V) from apower supply, which creates an electric field (E) to drive holes orelectrons, depending on the polarity of the bias applied to ITO. Al electrodecollected the carriers, which contribute to the current (I) that beingrecorded by an oscilloscope with a resistor for observing the photovoltagetransient signal. By reading the transit time (tT) from thetransient signal, the carrier mobility can be determined using the followingequation:Comparisons of the hole transient signals for the 2- and 0.3-μmNPB without and with the CGL. Under the photoexcitation, excitons weredissociated within the SubPc:C70 CGL and generates holes andelectrons that are transported by SubPc and C70 molecules,respectively. To measure the hole transient signal, a positive bias was appliedto the ITO electrode, photogenerated holes were swept through the NPB layer andreaches the Al electrode. 57374962795582t1/2 is the time that the photovoltage drops to the halfof the value of the transit time (tT). A lower Windicates a less spread of the carrier transport through materials, thus leadingto an abrupt drop in the tail section instead of a slow descent. In general, themain idea of estimating the W parameter is for observing the effects oftraps on molecularly doped materials because carrier spread was brought about bythese trap states that may dominate the dispersivity of materials5582W was in a range of 0.25-0.30. This valueon average is twofold higher than that reported in previous studies thatobtained the value of W to 0.13 for neat NPB5758W decreased to less than 0.10 on average, which waslower than the value reported previously, thus indicating a more non-dispersiveproperty. This result was attributed to the well-confined charge-generationwidth as will be discussed in detail later. However, when the thickness of theNPB decreased to 0.3 μm, the transient signal wasdispersive and the transit time is indeterminable without using the CGL, asshown in W values slightlyincreaseed from 0.07 to 0.12 on average, which is similar to the value obtainedfrom the NPB with the thickness of 7-10 μm under anexcitation of 337 nm. Because of the different absorption propertiesbetween the CGL and the material under test , we reduced the TOF sample thickness to0.3 μm, which is the thinnest value for determining thehole mobility of the NPB to date.where et al.,power-dissipation profiles enabled predicting optical-field distribution andpower dissipation in thin-film based devices5758722447484950535570mixture which absorbs an incident wavelength of 532 nm and providesa well-confined charge-generation width of less than 100 nm, asshown in 535860To determine the reduced degree of dispersion when the CGL was used, we proposeda new approach to characterize the generation width of photoexcited carriersthat is crucial for contributing to the dispersivity of the transient signal. Onthe basis of the transfer matrix method as proposed by Pettersson 3 (15 nm)/NPB(150 or 300 nm)/Au, detertemed using a SCLC model is provided . For measuring thecarrier mobility of polymers, the discussed structure can be altered toITO/polymer/CGL/Al to resolve the dissolving problem. To demonstrate this idea,a structure of ITO/NPB (0.3 μm)/CGL(10 nm)/Al was fabricated for comparison with ITO/CGL(10 nm)/NPB (0.3 μm)/Al. Because the CGL ison the top of the NPB, the measurement setup is different from the case in whicha CGL is underneath the NPB . Figure 188990919293The TOF measurement is widely used to evaluate the carrier mobility of organicmaterials over the last decade. To obtain a plateau and an abrupt drop of thetransient signal, the sample thicknesses were typically several micrometers.However, in practical electronic devices the thickness is between tens to hundredsnm, or sub-micrometers. The requirement of a thick TOF sample is primary due to anunwell-confined charge-generation width. This requirement could have resulted inoverestimating carrier mobility because the travelling distance of carriers is notequivalent to the thickness of materials under test, especially parts of carrierstravel a shorter distance. Although the concept and the use of a CGL for confiningthe charge-generation width is not new, concerning that both small-molecule andpolymeric CGLs have been reported245970 unintentionally gave rise to a P3HT/C70bilayer OPV structure, the hole mobility showed consistent results independent ofthe illuminating direction  was not limited to measure the carrier mobility of smallmolecules but also polymers by simply changing the sample preparation method.In summary, we used OPV materials, SubPc and C3, SubPc, C70, BCP, NPB, Al werepurchased from Sigma-Aldrich. The SubPc was purified twice prior to use, whereasthe rest of the organic materials were used as received. ITO-coated glasssubstrates were cleaned with ultrasonic bath in solutions  without further surface treatment, either for OPVdevices or TOF samples. The thin film deposition was conducted in a high-vacuumchamber(<8 × 10−6 Torr).The deposition rates and film thicknesses were monitored during the depositionusing a deposition controller (Sycon Instruments XTM-2XM) with a quartz crystalmicrobalance. The film thickness of each layer was measured using a surfaceprofiler (Veeco Tektak 3) and an ellipsometry (Raditech SE-950). The depositionrate of each layer in the OPV devices was controlled at approximately0.1 nm/s. To form a SubPc:C70 mixture in a volume ratioof 1:5, the deposition rates of the SubPc and C70 were independentlycontrolled to approximately 0.04 and 0.20 nm/s, respectively. Forthe TOF samples, NPB layers were deposited at 0.3-0.5 nm/s, for both2 and 0.3 μm. The deposition of Al cathode through ashadow mask defined the active area of 0.04 cm2 forboth the OPV devices and TOF samples. After the cathode deposition, the deviceswere appropriately encapsulated using an UV-curable epoxy resin  and a getter-attached cover glass for preventing theorganic layers from the ambient environment. A home-made in-situand independently controlled shutter system in which each shutter can beindividually controlled was used to fabricate the devices with variousstructures in one run without breaking the vacuum, thus preventing the deviationbetween the production runs. A home-designed layout on the ITO substratesenabled producing five devices on one substrate. A total number of fifteendevices were fabricated simultaneously on three different substrates.All materials, MoOJ-Vcharacteristics were measured using a source meter (Keithley 2636A). PhotoJ-V characteristics were performed under AM 1.5G solarillumination (Newport 91160A) at100 mW cm−2 which wasmeasured using a silicon reference cell . The EQE spectra were determined usinga monochromator (Newport 74100) for generating monochromatic light in awavelength range of 400 to 800 nm and a lock-in amplifier (StanfordResearch Systems SR830) chopped at 250 Hz for recording thephotocurrent from the OPV devices.All characterizations were performed in the air. HOMO levels and work functions of thin films were evaluated from high-energyonset points in a spectrum measured using a photoelectron spectrometer (RikenKeiki AC-2). Energy gaps were derived from the absorption spectra by thehigh-wavelength onset points recorded using a UV-visible spectrophotometer(Thermo Scientific Evolution 220) for estimating LUMO levels of the organicmaterials. Optical constants, refractive indices and extinction coefficients,were determined using the ellipsometry that was used in the measurement of filmthickness. EL spectra of laser emission were measured using a spectrometer(Ocean Optics USB2000+).An Nd:YAG pulsed laser (Spectra-Physics Quanta-Ray INDI-40-10) with second andthird harmonic wavelengths, 355 and 532 nm, respectively, were usedas excitation sources. The pulse width was in a range of 6-9 ns. The power of355- and 532-nm laser was approximately 20-30 mW measured using apower meter (Scientech AC2501). The laser beam diameter was approximately10 mm, which covered the entire area of the TOF samples. The sourcemeter that was used in characterizing OPV devices provided the electric fieldacross the TOF samples for driving photo-excited carriers. The transient signalswere recorded using a 2.5 GHz oscilloscope (LeCroy WaveRunner 625Zi)triggered synchronously with the Q-switch Nd:YAG laser. For the NPB TOF samples,all excitation illuminated through the ITO side. For polymer TOF samples, theexcitation illuminated from both ITO and Ag sides.S.-W.L. and C.-C.L. contributed equally to this work. S.-W.L. and C.-C.L. providedthe idea of this research and wrote this paper. W.-C.S. and C.-H.Y. measured the TOFsamples and analyzed the data. C.-F.L., K.-T.C. and Y.-S.S. fabricated the TOFsamples. Y.-Z.L., T.-H.S. and Y.-H.L. summarized the references and provide theinformation about writing this paper. B.-Y.H. and W.-C.C. developed thehigh-performance OPV devices.How to cite this article: Liu, S.-W. et al. Downscaling the SampleThickness to Sub-Micrometers by Employing Organic Photovoltaic Materials as aCharge-Generation Layer in the Time-of-Flight Measurement. Sci. Rep.5, 10384; doi: 10.1038/srep10384 (2015).


Pseudomonas fluorescens switches to an alginate-producing phenotype when the pleiotropic anti-sigma factor MucA is inactivated. The inactivation is accompanied by an increased biomass yield on carbon sources when grown under nitrogen-limited chemostat conditions. A previous metabolome study showed significant changes in the intracellular metabolite concentrations, especially of the nucleotides, in mucA deletion mutants compared to the wild-type. In this study, the P. fluorescens SBW25 wild-type and an alginate non-producing mucA- ΔalgC double-knockout mutant are investigated through model-based 13C-metabolic flux analysis (13C-MFA) to explore the physiological consequences of MucA inactivation at the metabolic flux level. Intracellular metabolite extracts from three carbon labelling experiments using fructose as the sole carbon source are analysed for 13C-label incorporation in primary metabolites by gas and liquid chromatography tandem mass spectrometry.The bacterium mucA- ΔalgC mutant, while flux through the pentose phosphate pathway was increased. Our findings also indicated flexibility of the anaplerotic reactions through down-regulation of the pyruvate shunt in the mucA- ΔalgC mutant and up-regulation of the glyoxylate shunt.From mass isotopomer distribution datasets, absolute intracellular metabolic reaction rates for the wild type and the mutant are determined, revealing extensive reorganisation of carbon flux through central metabolic pathways in response to MucA inactivation. The carbon flux through the Entner-Doudoroff pathway was reduced in the mucA- mutant as a cell factory for alginate production would be to use non-growing conditions and nitrogen deprivation.Absolute metabolic fluxes and metabolite levels give detailed, integrated insight into the physiology of this industrially, medically and agriculturally important bacterial species and suggest that the most efficient way of using a The online version of this article (doi:10.1186/s12918-015-0148-0) contains supplementary material, which is available to authorized users. Pseudomonas is a bacterial genus containing species of industrial, agricultural and medical relevance due to their metabolic diversity and ability to colonise a wide variety of ecological niches, including not only soil and water but also insects, plants, and animals  mmol/(gDW h) of the MAL influx goes to PYR)  and normalised to fructose uptake for relative (B) comparison between the mutant and the wild type . On a relative scale, compared to the influx of the network  is 60% higher in the mucA- ΔalgC strain. Thus, the wildtype either uses more of the fructose for maintenance processes, has a less energy-efficient metabolism , and/or disposes of surplus energy through futile cycling, which was frequently described for other organisms such as Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum [Interestingly, the wild-type strain has a 60% higher fructose uptake rate than the  Figures . Becauseutamicum ,21-24.mucA- ΔalgC strain cannot be a direct effect of MucA inactivation alone, as an alginate-producing mucA- single-deletion mutant had a two-fold increased fructose uptake rate in the metabolome study (18.3 mmol C/(gDW h) in the mucA- mutant vs. 9.4 mmol C/(gDW h) in the wild type) [F6P and G6P alone.The reduction in fructose uptake in the ATP + 0.5ADP)/(ATP + ADP + AMP)) in both the alginate-non-producing mucA- ΔalgC mutant used in this fluxome study and an alginate-producing mucA- single-knockout mutant [ATP concentration and an increase in ADP and AMP concentrations, the latter almost 10 times higher. The higher EC of the wild type correlates with its larger NADH production . From this perspective, both P. fluorescens strains preferably use the less energy-efficient EDP route. Flamholz and co-workers analysed glycolysis and the EDP in terms of thermodynamic and kinetic constraints and concluded that glycolysis incurs a higher protein cost  than the EDP [In theory,  the EDP . The lowP. fluorescens is negatively regulated by AMP, as it is in other bacteria, e.g., E. coli [F6P to M6P, the induction of alginate synthesis in the mucA- mutant must alleviate the possible rate-limitation of the FBPase. The eight-fold increase in the mucA- mutants’ AMP concentration could have led to the direct conversion of FBP to DHAP and GAP by FBP aldolase (EC 4.1.2.13) in the mucA- ΔalgC strain, but that was not observed in the current fluxome study.Concerning the preferred route for fructose uptake, it is also interesting to note that FBPase in  E. coli . Because2, with energy conservation taking place through oxidative phosphorylation. This occurs with no obvious use of this extra energy compared to the mucA- ΔalgC strain. Based on current knowledge of co-factor dependencies [NADPH as well as NADH production are higher in the wild type  and Gram-positive (B. subtilis and P. versutus) bacteria [NADH production is not required.According to our fluxome study, a large proportion of the excess fructose uptake of the wild type is shuttled via EDP to TCA and complete oxidation to COndencies , the NADe Figure . Strikin species -29. The bacteria . Thus, tNADP if needed? Activation of a transhydrogenase (EC 1.6.1.1) may provide an explanation to this question [mucA- ΔalgC strain, it is unlikely that this mutant is energy limited. It is puzzling that the mutant activates the GLX shunt, which is mostly associated with growth on two-carbon substrates, and generates one net OAA from acetyl-CoA without any energy conservation. Additionally, and similarly to the wild type, the ANA cycling of the mucA- ΔalgC strain through PEP carboxylase (EC 4.1.1.31) and PEP carboxykinase (EC 4.1.1.49) could be regarded as a futile cycle because one net ATP is consumed. However, the extent of futile cycling could not be quantified because, as mentioned, ANA reactions were not resolved in the forward and backward directions with statistical confidence. To this end, further CLEs with specially designed 13C-label mixtures are needed [Yet there remain open questions: how does the cell regenerate or allocate question . Due to e needed ,31.The comparative investigation of changes in the transcriptome  and the mucA- ΔalgC strain and the glyoxylate shunt was activated, the pool sizes of the TCA metabolites were not altered, except for the pool size of succinate, which increased by a factor of 2.4 . Cra is a global sensor and regulator of primary metabolic fluxes in Gram-negative bacteria, with a dual role as both a transcriptional repressor and an activator [E. coli cra- mutant, indicating that Cra represses these pathways [F1P is the preferred effector of Cra in P. putida, and FBP is probably a minor effector as well [F1P mediated by Cra, or directly through MucA inactivation, was observed in P. fluorescens [F1P pool changes and metabolic flux reorganisation. Hence, metabolome and fluxome data provide a “true” orthogonal view of the metabolism of P. fluorescens, in the sense that understanding how cells modulate metabolic fluxes in response to interventions requires integrative multi-omics studies.Additionally, and not surprisingly, direct comparison of changes in the metabolome and the fluxome do not show any clear correlation: while the net flux through TCA was reduced by 50% in the ctivator ,33. For pathways . F1P is  as well . No stromucA- ΔalgC double-knockout P. fluorescens mutant was compared to the wild-type strain at the metabolic flux level to illuminate the global metabolic response to MucA inactivation on central carbon metabolism without a biasing carbon re-routing by alginate production. Corroborating transcriptome and metabolome results from former studies, a strong influence of the mucA- ΔalgC double-knockout mutant was shown, involving fine-tuned and significant reorganisation of carbon utilisation. Salient alterations lead to a decrease of the overall fructose uptake and an activation of the PPP and the GLX shunt that is accompanied by a deactivation of the EDP. Although both strains produce a similar amount of energy (NADH and ATP) per mole of substrate, the mutant seems to exhibit more energy-efficient behaviour than the wildtype.In this work, a mucA- mutants need to be further investigated. These experiments bear the potential to elucidate the complex control mechanisms of MucA on alginate production and central carbon metabolism. These control points may become important targets for drug development to prevent the formation of the mucoid phenotype, which leads to pulmonary infection in cystic fibrosis patients, or to increase industrial alginate production. This requires extending the multi-omics characterisation to further mutants, including catabolite repressor/activator cra mutants, and experimental conditions, i.e., growth rates and carbon sources, that are clinically and industrially relevant. From the modelling point of view, the application of non-stationary 13C-MFA in combination with advanced experimental design strategies would be advantageous to advance the integrative analysis of metabolite levels and fluxes and increase the statistical significance of the results, particularly with regard to anaplerosis.Clearly, to mechanistically elucidate the metabolic control mechanisms of alginate production on fluxes, P. fluorescens SBW25 and P. fluorescens SBW25 mucA-ΔalgC. Construction of the mutant strain was previously presented in detail [mucA-ΔalgC strain to look for effects of MucA inactivation other than alginate production is that it produces all biosynthetic enzymes required for alginate production other than AlgC.The strains used in this study were n detail  for the mucA- ΔalgC strain, respectively. These carbon source concentrations lead to a fructose concentration of 2.5 g/L or above in the fermenters at steady state. In brief, the chemostat cultivations were nitrogen limited using fructose as a carbon source with a dilution rate of 0.04 h-1. Temperature was maintained at 25°C, pH was maintained at 7.8 by automated sodium hydroxide addition, air flow was 1.5 VVM, and oxygen saturation was maintained above 20% by adjustment of the stirring speed. Cultivations were operated as batches for 24 h before initiating continuous operation with a feed containing naturally labelled fructose. After 1.5-2.0 volume exchanges, the feed stock was changed. Afterwards, the feedstock contained 13C-labelled fructose  and isotope enrichments of 99% (mole/mole); and one lot of U-13C-fructose with chemical purity of 100% (w/w) and isotopic enrichment of 99.5% (mole/mole), Cambridge Isotope Laboratories, Inc, Andover). Sampling was performed after an additional 3 volume exchanges. Process data of double knock out mutant is given in Additional file Chemostat cultivations were performed as described earlier , except 13C-labelled feed. In the first cultivation, the wild-type strain was grown using a 60.04% 1-13C-, 20.31% U-13C- and 19.65% 12C-fructose isotopomer composition . In the two main cultivations, the wild type and the mucA-ΔalgC strain were grown with the designed fructose isotopomer mixture of 60.08% 1-13C-, 39.92% U-13C-fructose.Three cultivations were performed; the only difference between the first and the two last was the fructose isotopomer composition used in the 660 and residual fructose measurement. The CO2 concentration in the off gas was measured with a Rosemount Binos 100 CO2 analyser. These data were used to calculate the carbon balance as described previously [Immediately prior to sampling for metabolite extracts, an aliquot of 5 mL was taken from the culture for ODeviously , except P. fluorescens SBW25 wild-type and the mucA-ΔalgC strains. Prior to LC-MS/MS analysis, samples were reconstituted in 500 μL 60% (v/v) methanol. Analysis was performed on an Agilent 1200 series LC connected via an electrospray ion source to an Agilent 6410 triple-quadrupole MS using an adaptation of the reverse phase tributylamine ion exchange method introduced by Luo et al. [P. fluorescens [n-m + 1)(m + 1) MRM-transitions for each compound, where n is the number of carbons in the precursor ion and m is the number of carbons in the product ion, respectively [Metabolite extracts were analysed by an LC-MS/MS method and a GC-MS/MS method to generate mass isotopomer distribution datasets for the o et al. . The samorescens . A totalectively . A list Samples for GC-MS/MS analysis were derivatised prior to analysis as described previously  using anmucA-ΔalgC, respectively. For mass isotopomers detected by the GC-MS/MS method, these STD values were 10%, 12% and 15%, respectively.The resulting mass isotopomer fractions are listed in Additional file P. fluorescens SBW25 was derived from the genome-scale metabolic model iSB1139 reconstructed by Borgos et al. [http://www.genome.jp/kegg). Construction of our model was guided by 1) representing degradation pathways by effluxes from the system and excluding low-abundance biomass components .A biochemical reaction network of central carbon metabolism of P. fluorescens central carbon metabolism consists of routes for fructose uptake (Carbon Uptake), EMP, EDP, PPP, TCA cycle including the GLX shunt, the ANA section, as well as the biomass equation, which defines the net fluxes of metabolites into amino acid and fatty acid synthesis. To formulate the latter effluxes, the biomass composition for P. putida devised by Nogales et al. [P. fluorescens wild type and the mucA-ΔalgC mutant strain.The model of s et al.  was adop13C-MFA protocols, we refer to classical review papers, e.g., [The generation of isotopomer balances, the simulation of measurements, the experimental mixture design, the flux estimation , and the statistical assessment of resulting flux confidence intervals were performed using the software tool 13CFLUX2 . For furs, e.g., . Finallys, e.g., .P. fluorescens SBW25 wild type. The main CLEs for the wild type and the mucA-ΔalgC strain were performed using the calculated OED mixture consisting of 60.08% 1-13C-, 39.92% U-13C-fructose isotopomers. When inspecting the data thoroughly with the isotopomer network at hand, some peak measurements have to be excluded due to low peak quality. Specifically, we used the following procedure to achieve an acceptable fit: 1) we assumed a minimum STD of 1% to account for the noise of measurements close to the detection limit of the MS instruments, 2) we obtained the best fit by minimising the difference between simulated and measured data, and 3) we excluded those measurements with low abundance that have extraordinary large discrepancies  study to determine an information-optimised fructose label mixture was performed based on the fluxes estimated from the pre-CLE with


Circulating microRNA-21 (miR-21) is known to be aberrantly expressed in hepatocellular carcinoma (HCC) patients, and this implies that microRNA-21 is a promising and novel indicator of HCC. However, a systematic evaluation of the performance of microRNA-21 as a diagnostic marker for HCC has yet to be conducted. Therefore, the test performance of circulating miR-21 for HCC was assessed in this study.Three common international databases and a Chinese electronic database were used to search for literature on the diagnostic accuracy of microRNA-21 for HCC. The pooled results included the sensitivity and specificity of microRNA-21 for HCC detection and were analyzed with a random effect model. The area under summary receiver operating characteristic curve (AUC) was used to estimate overall test performance.A total of 339 HCC patients and 338 controls without HCC from four published studies were eligible for the meta-analysis and included in our study. The test performance of circulating miR-21 in HCC detection was assessed with the summary estimates of sensitivity and specificity, which were 81.2% (95% CI: 70.8% to 88.4%) and 84.8% (95% CI: 75.1% to 91.2%), respectively. The value of AUC was 0.90 (95% CI: 0.87 to 0.92). Significant inter-study heterogeneity was detected by our analysis, and sub-group analyses suggested that the type of control group was probably a source of heterogeneity.Our current findings suggested that circulating miR-21 can serve as a potential co-biomarker for early-stage HCC diagnosis. Thorough large-scale studies are needed to confirm the generalizability of our findings. Primary liver cancer is the fifth commonest malignancy and its mortality ranks second among all cancer-related deaths worldwide . In 2012There are other common biomarkers of HCC, such as des-γ-carboxy prothrombin (DCP), glycosylated AFP-L3 fraction of total AFP and glypican 3 (GPC-3). Unfortunately, none have been found be specific of early-stage HCC . ConsideMicroRNAs (miRNAs) are small (~22 nt) non-coding RNAs that have vital functions in various cellular biological processes, including cell proliferation, differentiation, and apoptosis . It has MiRNA-21(miR-21) is one of the first oncogenic miRNAs with upregulation detected in many types of human cancer –19. In rA literature search  was performed on three international electronic databases  and a Chinese database . No limitations were imposed on language. The following two sets of keywords were used to retrieve relevant studies: (a) keywords for the primary disease: “hepatocellular cancer” or “hepatocellular tumor” or “hepatocellular carcinoma” or “hepatocellular neoplasm” or “liver cancer” or “liver tumor” or “liver carcinoma” or “liver neoplasm” or “HCC” and (b) keywords for the diagnostic biomarker: “microRNA-21” or “miRNA-21” or “miR-21” or “hsa-miR-21.” Our literature searches were limited to human studies. To validate the qualified studies, we also manually searched the references of review papers and other relevant studies.All publications were carefully assessed by two authors (ZTW and QBL). The third reviewer (JFY) helped resolve discrepancies through discussion with the first two authors. The criteria for inclusion were as follows: (a) histopathological examination was used as the gold standard for HCC diagnosis; (b) the diagnostic studies employed circulating miR-21 to diagnose HCC; (c) the studies had sufficient data to calculate the number of true positive (TP), false positive (FP), false negative (FN), and true negative (TN) results; (d) serum and/or plasma samples were collected before surgery and chemotherapy; (e) each individual study involved two treatment groups, namely, the HCC and control groups.In addition, the following exclusion criteria were used: (a) non-original papers, such as conference abstracts, letters, and reviews; (b) duplicate studies; and (c) studies without qualified data or with 20 patients or less.Data were independently extracted by two authors (ZTW and QBL) from the included studies. The following data were obtained: (a) study characteristics ; (b) characteristics of the subjects ; (c) sensitivity, specificity.The quality assessment of each included study was performed with Quality Assessment of Diagnostic Accuracy Studies (QUADAS) . This toP < 0.05 denoted statistical significance. The between-study heterogeneity was evaluated with the I2 index and χ2 test, such that a value of I2 > 50% and/or a p < 0.05 indicated heterogeneity [Meta-DiSc 1.4 and Stata 12.0 were used to perform all statistical analyses . P < 0.0ogeneity . A randoogeneity .The literature search identified 1,828 relevant articles, 207 of which were excluded as duplicated publications. After a preliminary review of titles and abstracts, a total of 1,612 articles  were excluded for various reasons. The full-text articles of the nine remaining publications were obtained, and five studies were excluded for insufficient data. Finally, four publications were selected for our meta-analysis –26. The The four selected publications described six eligible studies and were published from 2011 to 2013; the data involved 339 HCC patients and 338 healthy or chronic hepatitis controls. Three of the articles were published in English, whereas the study by Qin et al. was in Chinese . Histopa2 = 11.255, p = 0.002; I2 = 82.23%). Therefore, a random effects model was selected. The pooled sensitivity and specificity of circulating miR-21 for HCC detection is summarized in I2 = 89.65%) and specificity (I2 = 75.94%) results.Heterogeneity was present in our meta-analysis, as confirmed by the results  indicated the absence of the threshold effect in our meta-analysis.Threshold effect occurs when differences in sensitivities and specificities arise; this effect can be assessed with the Spearman correlation coefficient . A valuep = 0.78) and gender . Therefore, age and gender were probably not the sources of heterogeneity, and others factors might have caused the observed heterogeneity.With the exception of variances originating from the threshold effect, heterogeneity can be caused by other factors, such as different clinical or socio-demographic characteristics and differences in the study design . Meta-reI2 = 0.0%, p = 0.651), a result suggesting that the type of control group may be the source of heterogeneity. However, the pooled sensitivity and specificity of circulating miR-21 for discriminating HCC from chronic hepatitis were 62.4% (54.9% to 69.5%) and 84.4% (75.3% to 91.2%) based on the results for 181 HCC and 90 individuals with chronic hepatitis [The results from 282 HCC patients and 189 healthy individuals in three studies –24,26 wep-value of 0.856 suggested the absence of publication bias , and the value of the Spearman correlation coefficient was 0.100 (p > 0.05), which indicated the lack of heterogeneity caused by the threshold effect. Meta-regression analyses suggested that age and gender may not be the potential sources of heterogeneity among the studies, whereas the sub-group analyses suggested that the type of control group was probably a source of heterogeneity. Although the included studies have all been published, these studies originated from a small group of authors. Therefore, the reliability of the conclusions of our meta-analysis may be affected by publication bias. However, the funnel plot showed the absence of publication bias.Significant heterogeneity was noted in this meta-analysis Click here for additional data file.S1 Table(DOC)Click here for additional data file.


The purpose of this study was to evaluate the coherence between three different methods assessing the power driven from a counter movement jump (CMJ); the Powertimer 300-series contact mat (C-mat), the MuscleLab 4010 infrared mat (IR-mat) and the MuscleLab 4010 linear encoder (M-encoder), and to evaluate the test-retest reliability of the M-encoder.Twenty-two males and 29 female, elite athletes performed two test sessions with three days in between. Each test session included counter movement jumps (CMJ) performed on a Smith-machine with external loads of 40 kg. Jump height and flight time were assessed with C-mat and IR-mat, and power was additionally assessed with C-mat. Variables analyzed from the M-encoder were average power (AP), average force (AV), average velocity (AV), and distance (D).p = 0.95-0.98). The results showed a high test-retest reliability for all indices assessed with the M-encoder, AP , AF . Furthermore, the AV had high values  as well as D .The results from the C-mat were systematically higher than the ones obtained from the M-encoder and IR-mat. The correlation between the C-mat, M-encoder and the IR-mat was strong (rIt is important to use the same equipment in both pre- and post-testing, since all three methods were reliable, coherent but not interchangeable to each other. In sport science, aiming at enhancing elite athlete performance through exercise-training, accurate methods for testing the performance are important . PerformGenerating high power is important for many elite athletes and the use of loaded vertical jumps as an exercise-training method has been shown to be effective to increase muscular strength and power ,4. VertiEquipment widely used for testing muscular strength, power, and jump height are different types of contact mats ,10,11, iSeveral studies involving coherence, reliability, and CMJ have been conducted on contact mats and force platforms ,7,9. HowFifty-one individuals were recruited into this study. All of the subjects were team members of different teams at the highest leagues in Sweden, and thereby considered to be elite athletes. The participants consisted of athletes from football, basketball, volleyball, ice hockey, handball, and track and field sports. All participants were given an oral and written description of the test and signed an informed consent. Ethical principles outlined in the declaration of Helsinki were followed. This study was approved by the Ethics Committee in Lund, Sweden (ETIK 2009/699).Participants in the coherence analysis (session 1) were 22 men and 29 women. The men’s age, mass, and height were 22.5(4.7) years, 83.8(13.9) kg, and 184(8) cm, respectively. The women’s age, mass, and height were 20.3(3.2) years, 68(7.4) kg, and 174(6) cm, respectively. The external loading of 40 kg was 50(8) % and 59(6) % respectively.Participants in the test-retest analysis (session 1 and session 2) were 18 men and 23 women completed the tests on both test session one and two. The men’s age, mass, and height were 21.8(4.3) years, 81.9(13.7) kg, and 183(8) cm, respectively. The women’s age, mass, and height were 20.3(3.2) years, 68(7.4) kg, and 173(6) cm, respectively. The external loading of 40 kg was 50(8) % and 59(6) % respectively.−1 body weight . This was followed by a familiarization session of three jump trails at a sub-maximal level. During testing, verbal encouragement was used for all persons for each attempt. Two investigators administered all tests, but were responsible for different tasks and equipment in a standardized fashion.Test-retest CMJ with external load was performed on a Smith machine  with a three-day interval between tests to evaluate the test-retest reliability. The participants were informed to refrain from eating, drinking coffee, or smoking two hours before each test session. They were also informed to refrain from performing any heavy exercise 48 hours prior to the tests. Before testing, each person answered questions concerning their health and training status. Then they were weighted on an electronic glass scale  before they performed a ten-minute sub-maximal warm-up on a bicycle with a workload of 1 W∙kgAfter the three jump trails of familiarization, the test procedure started. The different loads for the men were 20, 40, 60, 80 and 100 kg and for the women 20, 30, 40, 50 and 60 kg. Each subject performed three jump trails on both legs with a barbell on their shoulders connected to a Smith machine. A three-minute rest followed the three jump trails on each load. The load was chosen because it is a common load used in our lab during testing of elite athletes. 40 kg was chosen to analyze the reliability and coherence at the same absolute external load for both men and women. Before the actual test started, the participants were told to bend their knees to about 90 degrees, which was marked on the Smith machine and measured with a conventional goniometer. At the same time, marks were placed for the hand and foot positions. During the CMJ, the participants completed a fast downward movement followed by a fast upward movement when the barbell reached the marking on the smith machine. The participants were given verbal guidance concerning the positions. The three trails during test session 1 were used in the intra-session analysis and the best jump was used for the evaluation of the inter-session and the coherence analyzes. The equipment used for measurement during all jumps was the M-encoder, IR-mat, and C-mat. M-encoder measured the average power (AP). IR-mat measured jump height and flight time. C-mat measured power, jump height, and flight time. The M-encoder was used for measures of average power (AP), average force (AF), average velocity (AV), and distance (D). To be able to analyze the knee angle in Dartfish , the jumps were recorded from the left side with a digital video camera . To make the analyses more convenient, tape markings were placed on trochanter major, the lateral condyle and just above the lateral malleolus of the fibula on the left leg.C-mat  a contact mat assessing flight time from when the subject’s foot leaves the contact mat until the foot touches the mat again. The jump height was calculated as formula jh = (g∙tf)2/8  and power was calculated as P = 60.7∙jh + 45.3 ∙bm-2055  using the software handed by the manufacturer.IR-mat  an infrared mat assessing flight time from when the subject’s foot leaves the infrared beam until the foot crosses the beam again. Jump height was calculated by the software handed by the manufacturer using the formula jh = (g∙tf)2/8 .M-encoder  a linear encoder assessing speed and acceleration of the barbell through a wire attached to the barbell. Average power was calculated by the software handed by the manufacturer using the formula P = F∙v . Average force was calculated by the software handed by the manufacturer using the formula F = m∙g + m∙ a .The Kolmogorov-Smirnov test was used to test the normal distribution of the data. Values throughout are given as means and standards deviations (SD). Comparing the results between session 1 and 2 for the different gendes, a 2 (gender) x 2 (session) analyzes was used with a repeated measures ANOVA approach. The gender (n = 2) and the sessions (n = 2) were considered as the within-participant factor. Greenhouse-Geisser correction was used and the Sidak adjustment was applied during the post hoc analysis for multiple comparisons.Intra-session reliability 2 (methods) x 3 (trails) was analyzed with a repeated measures ANOVA approach. The methods (n = 2) and the trials (n = 3) were considered as the within-participant factor. Greenhouse-Geisser correction was used and the Sidak adjustment was applied during the post hoc analysis for multiple comparisons. The choice of statistical approach was in agreement with Marina and Torrado .p), the ICC and CV were used to analyze the inter-session reliability. As part of the inter-session reliability analysis, the standard error of the measurement (SEM) was assessed and calculated as SEM = SD × √(1-ICC) [Test-retest correlations were calculated both for intra-session and inter-session relations. Intraclass correlation coefficient (ICC) and coefficient of variance (CV) were used to analyze intra-session reliability. The highest flight time, jump height and power were used for inter-session analysis. Pearson’s correlation (r√(1-ICC) . To anal√(1-ICC) . Also th√(1-ICC)  was calc√(1-ICC)  was calcThe following methods for assessing the coherence between M-encoder, IR-mat, and C-mat were used: 1) Mean difference and standard deviation with a 2 (methods) x 3 (trails) repeated measures ANOVA approach to detect statistical significant differences; 2) Pearson’s correlations were used to analyze strength of associations between methods; 3) M-encoder, IR-mat, and C-mat were compared two and two in a Bland-Altman analysis to find any systematic variance . The p <Ethical principles outlined in the declaration of Helsinki were followed. This study was approved by the Ethics Committee in Lund, Sweden (ETIK 2009/699).All data, except for power assessed with the M-encoder on the second session p = 0.014), were normally distributed according to the Kolmogorov-Smirnov test. Since no main effect was seen by session between the men and women  for all variables and loads. The assessments of AP show that there was a significant and strong correlation  between session 1 and 2. TE%, which is a value of the relative spread or ME, was 3.9%. The correlation and comparison between session1 and session 2 for AF assessed with M-encoder . The value for TE% (1.4%) was low. There was a strong and significant correlation  for assessments of AV between session1 and session 2 . TE% for D showed the highest values, of 5.4%. Knee angle had the lowest relation between session 1 and session 2  with a TE of 4.5%  was found  was between the C-mat and IR-mat for jump height and the Bland Altman plots demonstrated a mean difference of 2.7 cm with a LOA of 2.4 cm between assessments of jump height with C-mat and IR-mat. When comparing assessments of power with C-mat and M-encoder a significant relation was found  and a systematic bias with a mean difference of 2726 W and a LOA of 888 W.In the coherence analysis for flight time assessed with C-mat and IR-mat a significant relationship (rThe main finding of this study was that there is a high reproducibility of M-encoder, IR-mat and C-mat assessing CMJ performance among elite athletes. We have also shown that the power assessments obtained from the C-mat were systematically higher than the ones from the M-encoder.p > 0.9 as nearly perfect, 0.7-0.9 as very high, 0.5-0.7 as high, 0.3-0.5 as moderate, 0.1-0.3 as small, and 0.1 or less as trivial. In light of this, the correlation data in our study are nearly perfect or very high apart from the values for knee angle that was moderate (Tables Elite athletes need to test their progress of training on a regular basis and it is important that the test methodology is reliable and has a small within-subject variation  in ordere Tables .This study was designed to investigate the coherence of three different testing systems. The main equipment, the C-mat, was compared with both the M-encoder and IR-mat. There was strong coherence between both the C-mat and M-encoder and the C-mat and IR-mat. But, the difference obtained between the C-mat and M-encoder depends presumably on two different algorithms to calculate the power outcome. Also the construct of the contact mat and the encoder could presumably contribute to the differences between the two assessment methods. In contrast, the difference between the C-mat and IR-mat was more peculiar. The contact mat raised 6 mm above the ground and the IR-mat 16 mm above the ground, which could give a systematic bias since the starting point of assessment differs between the C-mat and the IR-mat. However, this does not explain the whole variation. Both the C-mat and the IR-mat used the same algorithm to calculate jump height, which is based on flight time. The flight time was measured by the C-mat from the moment the subject takes off from the mat to the moment the subject lands. The IR-mat flight time was measured from the moment the subject takes off and the infrared beam was switched off, until the subject lands again, at which time the infrared beam was switched on. The mean difference between the C-mat and the IR-mat was approximately 3 cm for jump height in our study, similar to the results obtained by Enoksen et al.  for jumpThe overall results show that the test-retest reliability was good, since the values for Pearson’s correlations coefficient and ICC between session 1 and session 2 were high. MDC%, TE% and LOA were low for the indices analyzed, except for MDC% of jump height assessed with C-mat Table . MeaningAtkinson and Nevill  discuss All the three assessment methods were reliable but not interchangeable. Assessments of flight time and jump height gave higher values assessed with the C-mat compared to the IR-mat in ms and cm respectively. Also the power assessments with the C-mat gave higher values compared to the M-encoder in watts.The results from the present study show that CMJ with external load assessed M-encoder is reliable. This knowledge will be of great interest to athletes and practitioners who use these tools. Athletes and practitioners will be able to carry out reliable tests and evaluate physical improvements, knowing that results are due to training and not due to variance in the test methodology.


A fault diagnosis strategy based on the wayside acoustic monitoring technique is investigated for locomotive bearing fault diagnosis. Inspired by the transient modeling analysis method based on correlation filtering analysis, a so-called Parametric-Mother-Doppler-Wavelet (PMDW) is constructed with six parameters, including a center characteristic frequency and five kinematic model parameters. A Doppler effect eliminator containing a PMDW generator, a correlation filtering analysis module, and a signal resampler is invented to eliminate the Doppler effect embedded in the acoustic signal of the recorded bearing. Through the Doppler effect eliminator, the five kinematic model parameters can be identified based on the signal itself. Then, the signal resampler is applied to eliminate the Doppler effect using the identified parameters. With the ability to detect early bearing faults, the transient model analysis method is employed to detect localized bearing faults after the embedded Doppler effect is eliminated. The effectiveness of the proposed fault diagnosis strategy is verified via simulation studies and applications to diagnose locomotive roller bearing defects. ApproxiHowever, the effectiveness of the wayside acoustic monitoring-based technique is decreased when vehicles pass by at high speeds. One of the problems caused by the high relative movement is the Doppler effect, as it can lead to obvious frequency shifts, frequency band expansion, and amplitude modulation for the recorded acoustic signal, which reduces the diagnostic performance .et al. ], a PMDW can be constructed through the following steps:(1)Discrete sine wave generation. The center frequency of the sine wave is cf. The discrete wave is an amplitude vector, eS, Amplitude modulation. Through the “Doppler-amplitude-modulator” implemented by rS, is obtained. The procedure is illustrated in (3)Amplitude vector rearrangement and curve fitting. rS obtained in Step 2 is rearranged by matching with the non-linear receive-time-vector, rt, which is calculated through the receive-time-calculator implemented by pχ, can then be obtained with rt and rS. The procedure is illustrated in (4)Signal resampling. The amplitude vector of a PMDW is then obtained by resampling the curve, pχ, obtained in Step 3 with the delayed-time-vector, dt, which is equal to et + 0/cR. R0 represents the distance between the starting point A and the microphone.Given an initialized model parameter set of γTwo PMDWs are generated through the preceding steps. The PMDW shown in 2.1.2.et al. , and I(n) is the imaginary part of A* [γΨ(n)], dopX̄ stands for the mean of dopX, R̄ and Ī stand for the mean of R and I respectively.The Pearson's correlation coefficients are calculated as follows:(3)x(n) and Ψ(n) is as follows:The criterion of the inherent linear relationship between Given an input Doppler-shifted signal, 2.1.3.After the kinematic model parameters are identified, the Doppler-shifted signal and the identified kinematic model parameters are inputted into the signal resampler, through which the embedded Doppler effect can be clearly eliminated.rdopS(n) with a length of dopN points, then the variables similar to those mentioned in Section 2.1.1 can be calculated as follows:If the identified kinematics model parameters are as follows:dopN equals to the length of the input Doppler-shifted signal.Emit-time-vector:dopR (n) equals:dopX(n) equals to Receive-time-vector:dopV(n) equals to According to dopθ (n) equals:and cosThe detailed procedure of the signal resampler is shown in kγ = ], the procedure of the resampler can be described as follows:(1)Curve fitting. The amplitude vector of the input Doppler-shifted signal is matched with the delayed-time-vector, ddopt, which equals  = [1a] = 40 m/s2. The wave form and FFT spectrum of the simulated Doppler-shifted bearing signal are illustrated in Through the PMDW generator introduced in Section 2.1.1, a sine wave can be embedded with the Doppler effect. If the sine wave is replaced with the aforementioned simulated signal, a Doppler-shifted bearing signal can be obtained with the same procedure and with the following kinematic parameters: The proposed diagnosis strategy is then applied to the Doppler-shifted bearing signal. The Doppler-shifted bearing signal is first inputted to the Doppler effect eliminator introduced in Section 2.1 to eliminate the embedded Doppler effect. Through the eliminator, the kinematic parameters are first identified through the correlation filtering analysis introduced in Section 2.1.2 between the input Doppler-shifted signal and the PMDWs constructed with parameter subsets described by fcS, is determined as [800:10:1200] by inspecting the FFT spectrum of the Doppler-shifted signal. The sound speed is set as cS = [320:1:360]. The parameter sets rS, X0S, and V0S are set as [0.5:0.1:1.5], [2:0.1:6] and [20:0.1:40], respectively. Only the first-order derivative of the moving speed, 1a, is considered, and aS is set as [35:0.5:45].The selection of the parameter subsets is crucial. On the one hand, the larger interval range and the smaller step of the parameter subset obtain a more accurate result. On the other hand, the larger interval range and the smaller step of the parameter subset cost excessive computation and decrease the efficiency of the method. When both accuracy and efficiency are considered, the subset, A searching grid of the model parameters is constructed based on the aforementioned six parameter subsets. Once a group of parameters is determined, the parameters are inputted to the PMDW generator introduced in Section 2.1.1 to generate a PMDW. The correlation filtering analysis introduced in Section 2.1.2 is then performed between the PMDW and the simulated Doppler-shifted signal. ddopt = , where dχ, can be obtained by fitting the amplitude vector of the input Doppler-shifted signal with ddopt. Through the receive-time-calculator described by rdopt, can be calculated with the emit-time-vector, edopt = . The two time vectors are shown in dχ, by rdopt, we obtain the receive-amplitude-vector, rdopS(n). Finally, the obtained rdopS(n) is rearranged by matching with edopt and then demodulated by The identified kinematic model parameters and the Doppler-shifted bearing signal are then inputted to the signal resampler introduced in Section 2.1.3 to eliminate the embedded Doppler effect. First, the amplitude vector of the input Doppler-shifted signal is matched with the delayed-time-vector, The wave form of the obtained Doppler-free signal is plotted as an overlay on the original bearing signal, shown in T, F, and Z. The range of T, F, and Z are set as , [800:10:1200], and { [0.005:0.001:0.03] ∪ [0.04:0.01:0.1] ∪ [0.2:0.1:0.9]}, respectively. The subset of Z is non-uniform to provide higher resolution at lower damping ratio values so that the efficiency of the method can be retained. The grid of the model parameters is constructed according to F and Z for each element from set T. When a group of parameters is determined, the transient model is constructed according to the procedures introduced in Section 2.2.1. The correlation filtering analysis introduced in Section 2.1.2 is then performed between the transient model and the input Doppler-free bearing signal. T. The optimal parameters cf = 1000 and ζ = 0.05 when T is set at 0.025 s.After the Doppler effect is eliminated, the transient model analysis method introduced in Section 2.2 is applied to the Doppler-free signal to detect the defect. The transient model is constructed according to T are shown in As a comparison, the simulated signal before the Doppler effect elimination is also analyzed via the transient model analysis method with the same model parameter subsets. The maximal correlation coefficients for the different elements from set 4.4.1.Two separate experiments were implemented to achieve the Doppler-shifted acoustic signals from defective locomotive roller bearings and verify the effectiveness of the proposed method. The obtained Doppler-shifted bearing acoustic signals are then analyzed by the proposed diagnosis strategy.In the first experiment, the acoustic signal of a locomotive roller bearing with a single localized defect was acquired by the test bench in The second experiment can be referred to the model illustrated in A single artificial crack with 0.18 mm width was set by the wire-electrode cutting machine on the outer and inner race, as shown in 4.2.The wave form of the Doppler-shifted bearing signal with the out-race defect is shown in fcS is determined as [800:10:1600] by inspecting the FFT spectrum in cS = [320:1:360], determined by the value of 343.2 m/s that stands for the sound speed in standard air atmosphere.The proposed diagnosis strategy is then applied to the filtered Doppler-shifted bearing signal. The signal is first inputted to the Doppler effect eliminator introduced in Section 2.1 to identify the kinematic model parameters and eliminate the embedded Doppler effect. During the kinematic parameter identification procedure, the subset c is determined by the properties of the medium of air. In the Earth's atmosphere, the chief factor affecting the sound speed is the temperature. In practice, it can be estimated through the value calculated by the following practical formula:The value of sound speed rS and X0S are set as [1.5:0.1:2.5] and [2:0.1:6], respectively, based on a previous coarse manual measurement. As the momentum of the car is large and the time duration of the pass-by movement is extremely short, only the first-order derivative of the moving speed, 1a, is considered. V0S and aS is set as [20:0.1:40] and [0:1:10], respectively, by considering the acceleration performance of the testing car. In parctice, the range of initial speed can also be estimated by the length of the recorded signal and the dimension of the vehicle.The parameter sets PMDWs are then generated with different parameters from the previously determined parameter subsets by the PMDW generator introduced in Section 2.1.1 to generate a PMDW. The correlation filtering analysis introduced in Section 2.1.2 is then performed between the generated PMDW and the Doppler-shifted signal. ddopt, which equals , where 0dopR equals dχ, can be obtained by fitting the amplitude vector of the input Doppler-shifted signal with ddopt. The receive-time-vector, rdopt, can be calculated from the emit-time-vector, edopt = , through the receive-time-calculator described by dχ, by rdopt to obtain the receive-amplitude-vector, rdopS(n). After the amplitude vector rearrangement by matching with edopt and the amplitude demodulation procedure implemented by The signal resampler introduced in Section 2.1.3 is then employed to eliminate the embedded Doppler effect with the identified kinematic parameters in The wave form of the obtained Doppler-free signal is shown in T = [50/50000:1/50000:600/50000], F = [800:5:2400], and Z = { [0.005:0.001:0.03] ∪ [0.04:0.01:0.1] ∪ [0.2:0.1:0.9]}. When a group of parameters is determined, the transient model is constructed according to the procedures introduced in Section 2.2.1. The correlation filtering analysis introduced in Section 2.1.2 is then performed between the transient model and the input Doppler-free bearing signal.The transient model analysis method introduced in Section 2.2 is then applied to detect the characteristic interval of the Doppler-free fault signal. A periodical transient model with parameters adjustable using T. The optimal impact period is 0.0072 s, which is equal to the real bearing fault-related impact interval. The optimal transient model is shown in The outer race characteristic frequency is 138.74 Hz as calculated by T are shown in The signal before the Doppler effect elimination is also analyzed via transient model analysis method with the same model parameter subsets. The maximal correlation coefficients for the different elements from set The obtained Doppler-shifted bearing signal  with an fcS is determined as [1400:10:2000] by inspecting the FFT spectrum in cS, rS, X0S, V0S, and aS, are set as [320:1:360], [1.5:0.1:2.5], [2:0.1:6], [20:0.1:40], and [0:1:10], respectively. PMDWs are then generated with different parameters from the determined parameter subsets by the PMDW generator introduced in Section 2.1.1 to generate a PMDW. The correlation filtering analysis introduced in Section 2.1.2 is then performed between the generated PMDWs and the simulated Doppler-shifted signal. The proposed Doppler effect eliminator is first applied to identify the kinematic model parameters and eliminate the embedded Doppler effect. The subset ddopt, which equals , where 0dopR equals dχ, can be obtained. Second, the receive-time-vector, rdopt, is calculated from the emit-time-vector, edopt = , through the receive-time-calculator described by dχ, by rdopt, the receive-amplitude-vector, rdopS(n), is obtained. Finally, the amplitude vector is rearranged by matching with edopt and performing the amplitude demodulation procedure through The embedded Doppler effect is then eliminated through the signal resampler introduced in Section 2.1.3. First, by matching the amplitude vector of the input Doppler-shifted signal with the delayed-time-vector, The wave form of the obtained Doppler-free signal is illustrated in T = [50/50000:1/50000:600/50000], F = [1200:5:2200], and Z = { [0.005:0.001:0.03] ∪ [0.04:0.01:0.1] ∪ [0.2:0.1:0.9]}. T.The transient model analysis method introduced in Section 2.2 is then applied to detect the characteristic interval embedded in the Doppler-free fault signal. A periodical transient model with parameters adjustable using The periodical impact interval is 0.0052 s, calculated by T are shown in The Doppler-shifted signal is then directly analyzed by the transient model analysis method with the same model parameter subsets. The maximal correlation coefficients for the different elements from set 5.In this paper, a fault diagnosis strategy based on the wayside acoustic monitoring technique is invented for locomotive bearing fault diagnosis. A parametric wavelet called PMDW is introduced and employed to identify the kinematic model parameters based on correlation analysis. A time domain signal resampler is introduced and employed to eliminate the embedded Doppler effect in the recorded bearing acoustic signal. The transient model analysis method is also employed to detect the localized bearing faults after the Doppler effect is eliminated. One of the best benefits of the proposed strategy is that all the kinematic model parameters, including the sound speed and the moving speed of the vehicle, as well as the geometric parameters of the model, can be identified based on the recorded signal itself. Thus, the proposed strategy overcomes the difficulties of kinematic model parameter measurement and is adjustable to different types of passing vehicles. Besides, the embedded Doppler effect can be eliminated through the proposed strategy, paving the way for the conventional invented signal processing methods and feature extraction methods. The performance of the proposed strategy has been evaluated by both simulated and practical Doppler-shifted bearing signals carrying fault information. Given the merits revealed in this study, the proposed fault diagnosis strategy can be widely used in wayside health monitoring systems, particularly in situations when vehicles pass by at high moving speeds and kinematic model parameters are difficult to estimate. The proposed data-driven Doppler effect eliminator is also hopeful to be used in other areas such as acoustic communication techniques and sound field holography for moving vehicles.


MicroRNAs (miRNAs) play important roles in regulating biological processes at the post-transcriptional level. Deregulation of miRNAs has been observed in cancer, and miRNAs are being investigated as potential biomarkers regarding diagnosis, prognosis and prediction in cancer management. Real-time quantitative polymerase chain reaction (RT-qPCR) is commonly used, when measuring miRNA expression. Appropriate normalisation of RT-qPCR data is important to ensure reliable results. The aim of the present study was to identify stably expressed miRNAs applicable as normaliser candidates in future studies of miRNA expression in rectal cancer.We performed high-throughput miRNA profiling (OpenArray®) on ten pairs of laser micro-dissected rectal cancer tissue and adjacent stroma. A global mean expression normalisation strategy was applied to identify the most stably expressed miRNAs for subsequent validation. In the first validation experiment, a panel of miRNAs were analysed on 25 pairs of micro dissected rectal cancer tissue and adjacent stroma. Subsequently, the same miRNAs were analysed in 28 pairs of rectal cancer tissue and normal rectal mucosa.P = 0.801, second validation P = 0.321). MiR-645 was excluded from the data analysis, because it was undetected in 35 of 50 samples  and in 24 of 56 samples , respectively. Significant difference in expression level of RNU6B was observed between tumour and adjacent stromal , and between tumour and normal rectal mucosa .From the miRNA profiling experiment, miR-645, miR-193a-5p, miR-27a and let-7g were identified as stably expressed, both in malignant and stromal tissue. In addition, NormFinder confirmed high expression stability for the four miRNAs. In the RT-qPCR based validation experiments, no significant difference between tumour and stroma/normal rectal mucosa was detected for the mean of the normaliser candidates miR-27a, miR-193a-5p and let-7g (first validation We recommend the mean expression of miR-27a, miR-193a-5p and let-7g as normalisation factor, when performing miRNA expression analyses by RT-qPCR on rectal cancer tissue. MicroRNAs (miRNAs) are short non-coding RNA molecules acting as negative gene regulators at the post-transcriptional level. These small RNAs consist of 18–25 nucleotides and play an important role in the regulation of biological processes such as cell differentiation, proliferation, and apoptosis. Changes in miRNA expression profiles are associated with abnormal cellular functions and have been observed in a variety of diseases, including cancer . BecauseReal-time quantitative polymerase chain reaction (RT-qPCR) is widely accepted as the method of choice for relative gene expression quantification, since it is the most sensitive and reproducible method. However, the accuracy and precision of the results are dependent on proper data normalisation. Reference genes are necessary to correct for non-biological sample-to-sample variations, which could be introduced during all steps from sample preparation to amplification. The use of non-stably expressed reference genes for normalisation may lead to incorrect conclusions. It is generally accepted that a universal reference gene suitable for all tissue types does not exist –8. The mDespite an increasing number of miRNA expression studies, the literature is extremely sparse, when it comes to rectal cancer and reliable normaliser candidates.The aim of the present study was to identify stably expressed miRNAs in rectal cancer tissue to be used as normalisers in future miRNA expression studies. In this context miRNA-normalisers will be referred to as reference genes. We studied the expression profile of miRNAs in laser micro-dissected tumour tissue and adjacent stromal tissue from specimens of patients with rectal cancer. First, we selected the most stably expressed miRNAs from our high-throughput technology based miRNA expression profiling study on 10 patients . Secondly, the stability of these miRNAs was further assessed by RT-qPCR in rectal cancer tissue from 25 other patients. Finally, we analysed the stability of the same miRNAs in rectal cancer tissue and normal rectal mucosa from 28 comparable patients. We included the commonly used small nuclear RNA RNU6B in all our analyses.Patients were randomly selected from a cohort of 239 patients undergoing resection of rectal cancer at Vejle Hospital, Denmark from 1999 to 2008. Inclusion was accomplished only if their histopathological diagnosis were rectal adenocarcinoma, if they had no preoperative chemo-radiotherapy, and if formalin-fixed paraffin-embedded (FFPE) rectal cancer tissue was available.For the miRNA expression profiling analysis, tumour tissue from a total of 10 patients  was selected representing different pathological tumor stages (pT1-pT4) and different amount of lymphocytes in the tumour micro-environment . For theAccording to The Danish National Committee on Health Research Ethics, ethical approval and written informed consent were not required, because the purpose of the study was quality development and no clinical data were further analysed . No samples were collected specifically for the purpose of this study. The samples were collected during standard treatment. The project was done anonymously. The tissue used in this study was confirmed not to be included in the Danish Registry of Human Tissue Utilisation. The study has been reported to the Danish Data Protection Agency.To specifically evaluate miRNA expression in rectal adenocarcinoma cells and in the surrounding stroma, laser micro-dissection (LMD) was performed . AvailabRNA was isolated using the miRNeasy FFPE Kit  according to the manufacturer’s instructions. Since paraffin was removed before LMD, first step was adding a lysis buffer containing proteinase K, followed by a short incubation at a higher temperature. This was followed by DNase treatment and RBC buffer treatment. Ethanol was added and the sample was applied to an RNeasy MinElute spin column, where the total RNA, including miRNA, bound to the membrane was washed away. Finally, total RNA, including miRNA, was eluted in 14 μl RNase-free water.OpenArray® Human MicroRNA Panel  is a high-throughput PCR-based miRNA array that enables analysis of more than 750 pre-defined miRNAs on a microfluidic platform. Single-stranded cDNA was reverse transcribed from total RNA using Megaplex™ RT Primers, Human Pool A or B. Each RT reaction had a final volume of 7.5 μl and contained total RNA (3 μl) and reagents from TaqMan® MicroRNA Reverse Transcription Kit (4.5 μl) (Life Technologies).Preamplification (16 cycles) was performed with Megaplex™ PreAmp Primers, Human Pool A or B (Life Technologies) and TaqMan PreAmp Mastermix (Life Technologies) according to the manufacturer’s standard instructions for low sample input (LSI). The PreAmp Product was diluted 1:40 in TE-buffer. To test the result of reverse transcription and preamplification before we proceeded to OpenArray®, the products were tested with single assay qPCR for either miR-16 (Pool A) or miR-151-3p (Pool B). Analyses were done in 20 μl reactions in duplicate using 1.3 μl of diluted sample. Both miRNAs were detected.For the qPCR step, TaqMan® OpenArray® Human MicroRNA Panels (Life Technologies) were used according to the manufacturer’s instructions. qPCR was performed using QuantStudio 12K Flex System (Life Technologies). All reactions were performed in triplicate, where each sample was analysed on three different OpenArray® Panels.The Custom TaqMan® Array MicroRNA Cards (Life Technologies) were used according to the manufacturer’s standard instructions for LSI. RNA was reverse-transcribed using Custom MiRNA RT Primer Pools supplied together with the Array Cards. Each RT reaction contained 3 μl total RNA and 4.5 μl RT reaction mix from TaqMan® MicroRNA Reverse Transcription Kit.5 μl RT product was preamplified (14 cycles) with Custom PreAmp Primer Pools (supplied with the Array Cards) and TaqMan PreAmp Mastermix in 25 μl reactions. The PreAmp Product was diluted 1:4 in TE-buffer. qPCR analyses were carried out using TaqMan® Universal Master Mix II NoAmpErase® UNG and the Applied Biosystems 7900 HT Real-Time PCR System. All reactions were performed in triplicate.Both validation experiments followed the same steps as described above.Cq−ΔΔ, where Cq is defined as the PCR cycle number at which the fluorescence meets the threshold in the amplification plot.High throughput data generated from TaqMan® OpenArray® RT-qPCR was analysed using Expression Suite Software Version 1.0.3 (Life Technologies), a data-analysis tool that utilises the comparative Cq (ΔΔCq) method to quantify relative gene expression across a large number of genes and samples. Relative quantification (RQ)–also called Fold Change (FC)—was calculated from Cq values according to the equations: (a) ΔCq = Cq (miRNA)–Cq ; (b) ΔΔCq = ΔCq (tumour)–ΔCq (stroma); and (c) RQ = 2Expression Suite performs an unpaired t-test for biological group comparisons, assuming that Cq values for both groups follow a normal distribution.Cq−, assuming that the amplification efficiency for the reference gene assays is close to 100%. Statistical analyses were performed using NCSS 2007 . In the validation experiments, Wilcoxon Rank-Sum Test was used to test for differences in medians, when comparing tumour and stroma  and tumour and normal mucosa . P-values below 0.05 were considered statistically significant for all tests.To analyse the expression stability of the reference gene candidates, the software program NormFinder  was usedWe profiled a panel of more than 750 miRNAs and controls on 10 pairs of rectal cancer tissue and adjacent stromal tissue. Initially, a global mean expression normalisation strategy was applied to identify the most stably expressed miRNAs . Data weFrom the Expression Suite data-analysis, the four reference gene candidates let-7g, miR-193-5p, miR-27a, and miR-645  were selTo add another generally accepted approach, a panel of 10 miRNAs, all meeting the above mentioned criteria ((a)-(d)), were analysed using Norm Finder . RQ rangP = 0.004) between the tumour tissue and the adjacent stromal tissue, whereas no significant difference was detected for the mean of the other three reference gene candidates: let-7g, miR-193a-5p and miR-27a (P = 0.801).The expression patterns of let-7g, miR-193a-5p, miR-27a, miR-645 and RNU6B were further analysed on 25 pairs of rectal cancer and adjacent stromal tissue. Because miR-645 was only detected in 15 of 50 samples, this miRNA was eliminated from further data analyses. Let-7g, miR-193a-5p and miR-27a showed different but measurable expression levels and all of them were expressed in all 50 samples. Raw Cq levels ranged from 20.1 to 28.9 for let-7g, from 23.2 to 31.1 for miR-193a-5p, and from 19.7 to 26.4 for miR-27a. For RNU6B the raw Cq level ranged from 12.0 to 23.6. Box plots are shown for the arithmetic mean of let-7g, miR-193a-5p and miR-27a, and for RNU6B in P = 0.321) between the tumour tissue and the normal mucosa, whereas for RNU6B, we observed a significant difference (P = 0.031).As a second validation, the expression patterns of let-7g, miR-193a-5p, miR-27a, miR-645 and RNU6B were analysed in 28 pairs of rectal cancer tissue and normal rectal mucosa. MiR-193a-5p, miR-27a and RNU6B were detected in all 56 samples. Let-7g was detected in 55 samples and undetected in one sample with normal rectal mucosa. Because miR-645 was only detected in 32 of 56 samples, this miRNA was not included in the further data analysis. Raw Cq levels ranged from 18.2 to 29.5 for let-7g, from 20.9 to 34.7 for miR-193a-5p, from 18.7 to 29.5 for mi-27a, and from 14.3 to 29.7 for RNU6B. Box plots are shown in The use of reliable reference genes for normalisation is of great importance, when performing RT-qPCR based research. The normalisation procedure compensates for variations in RNA-extraction yield, reverse-transcription yield, and efficiency of amplification, thereby enabling comparison of miRNA expression levels across different samples .It is widely accepted, that a single, universal reference gene does not exist, and it has been argued, that the most precise normalisation is achieved by selecting reference genes belonging to the same class of RNA as the investigated genes. Furthermore, it is recommended to search for stably expressed genes in each experimental system , 13.et al. reported the first systematic identification of reference gene candidates for miRNA RT-qPCR experiments in breast cancer in 2008 [et al. reported their identification of reference gene candidates for miRNA RT-qPCR in colorectal cancer [Davoren  in 2008 . In 2010l cancer , where tTo find particular information about reference genes for miRNA expression studies in rectal cancer, we performed a PubMed search. Using the MeSH terms “MicroRNAs” and “Rectal cancer”, we found 9 articles on rectal cancer expression studies, published until February 2015 that included information on the use of reference genes  on 10 pairs of rectal cancer tissue and adjacent stromal tissue. Since the reference genes are going to be used for normalisation in future RT-qPCR analyses on rectal cancer biopsies containing only tumour cells and adjacent stromal tissue, we did not include normal rectal mucosa in the initial analyses. By applying the global mean expression value for normalisation, we identified the most stably expressed miRNAs: let-7g, miR-193a-5p, miR-27a and miR-645. Global mean normalisation has previously been demonstrated to be superior to other approaches of normalisation in reducing technical variations and showing biological changes [Subsequent use of NormFinder confirmed high expression stability for the four miRNAs, which were all about ten times more stable than the often used reference gene RNU6B. Since the amount of stromal lymphocytes did no substantial alterations to the stability values, we concluded that the amount of lymphocytes did not influence the expression levels of these miRNAs, and the number of lymphocytes in the stromal compartment was not taken into consideration in the validation experiments.P = 0.801) in mean expression value for let-7g, miR-193a-5p and miR-27a between rectal cancer tissue and adjacent stromal tissue. The absence of a significant difference does not prove an equal expression level between tumour tissue and adjacent stromal tissue; however, on the other hand a high p-value speaks in favour of equality or minor differences. The significant difference in expression of RNU6B between tumour tissue and adjacent stroma (P = 0.004) is in concordance with previous results [Our first validation experiment was carried out in a larger cohort of 25 pairs of tissue and confirmed no significant difference P = 0.01 in meaP = 0.321) between the arithmetic mean of the expression levels of let-7g, miR-193a-5p and miR-27a in the rectal cancer tissue and the compared compartment , whereas a significant difference was detected for the expression level of RNU6B (P = 0.031). As in the first validation study, miR-645 was undetected in a considerable part of the samples (24 of 56), and therefore still not suitable as a reference gene in this setting.In order to further validate the results, we did a second validation in a cohort of 28 pairs of rectal cancer tissue and normal rectal mucosa. As in the two initial experiments, we did not find a significant difference Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Diabetes is probably responsible for worsening of metabolic syndrome (MetS)components. The aim of the present study was to compare the components of MetS between premenopausal and postmenopausal women with type 2 diabetes (T2DM).In this cross sectional study, we studied 639 women with T2DM that were divided in pre-menopausal (n = 221) and post-menopausal (n = 418) group. They were selected from participants of a diabetes clinic and assessed for MetS and its components. All MetS components were evaluated to follow age and duration of diabetes adjusted according to the ATP III criteria.The mean ages of pre-menopausal and post-menopausal were 43.33 ± 0.47 and 60.35 ± 0.38 years, respectively. MetS was defined for 88.3% of total subjects . Systolic blood pressure (SBP) and waist circumference (WC) were significantly higher in post-menopausal women with T2DM in comparison with pre-menopausal ones. There were no significant differences in triglyceride (T.G) level, diastolic blood pressure (DBP) and high density lipoprotein cholesterol (HDL-C) between the two groups. Myocardial infarction (MI) occurred in 1% total subjects (1.3% and 1.8%) in pre-menopausal and post-menopausal women with T2DM, respectively (p = 0.21).Worsening of MetS and its components except for SBP and waist circumference has been shown in pre-menopausal women with T2DM similar to post-menopausal ones. The observed differences may be explained by increasing age. With respect to increasing of myocardial infarction in premenopausal subjects, we suggest that diabetes can abolish the protective effects of premenopausal status for MetS and MI. Metabolic syndrome (MetS) is a cluster of characteristics related to insulin resistance that increase the risk of cardiovascular disease (CVD) and type II diabetes mellitus T2DM) . Global DM . GlobWe performed a clinical, cross-sectional study. The study population consisted of 639 participants, including 221 pre-menopausal women with T2DM and 418 post-menopausal women with T2DM. Patients were recruited from the diabetes clinic of Vali-Asr Hospital, which is affiliated with Tehran University of Medical Sciences.We defined menopausal status according to the definition of the Reproductive Aging Workshop (2011) . Pregnan2) was calculated according to the Quetelet formula. Blood pressure was measured with a standard calibrated mercury sphygmomanometer, applied to the right arm after 15 minutes of rest in the sitting position. In pre-menopausal subjects, 80.9% were being treated with one oral hypoglycemic agent and 19.1% received insulin therapy. In post-menopausal women, 75.4% received oral hypoglycemic agents and the other 24.6% received insulin as a glucose-lowering drug  criteria . As detaAll data were analysed separately for pre-menopausal and post-menopausal groups. For continuous variables, mean ± SD was calculated. Data analysis was performed with the statistical package for the social sciences program . Student’s t-test and analysis of variance considering age and duration of diabetes as covariates were used to compare the variables between the two groups. Adjustments were made for all independent variables. Odds ratios with 95% CI were calculated by binary logistic regression analysis for factors affecting MetS. All analyses were two-tailed and a P-value of less than 0.05 was considered statistically significant.MetS was defined for 88.3% of total subjects  (P = 0.34). Table Comparisons of the MetS components in pre- and post-menopausal groups are presented in Table The association of MetS components with menopausal status in women with T2DM is shown in Table The components of MetS, according to the ATP III Index, in pre-menopausal and post-menopausal women with T2DM are shown in Figure In the present study, the differences between the components of MetS in post-menopausal women compared with pre-menopausal women were questioned, as T2DM was the underlying condition. There was no significant difference in MetS prevalence in pre-menopausal and post-menopausal women with T2DM. In the present study, WC and systolic blood pressure differed significantly between pre-menopausal and post-menopausal women with T2DM. Diastolic blood pressure, triglycerides, HDL-C and FBS did not differ between the two groups.The frequency of MetS increases from the beginning of the menopausal transition to post menopause and the defined components of MetS are more frequent in non-diabetic, post-menopausal women compared to pre-menopausal women ,9. Ainy Diabetes is an inflammatory state, characterised by increased plasma levels of reactive oxygen species, lipid per oxidation products, CRP, ferritin and other reactive compounds, which all increase with inflammatory process ,11. FurtIn this study, HDL-C level did not differ in pre-menopausal compared with post-menopausal status in diabetic women. It has been proposed that in patients with T2DM, HDL is less effective at inhibiting oxidation and cholesterol efflux stimulation from macrophage ,14. In tIn the present study, WC differed between pre-menopausal and post-menopausal groups. Abdominal obesity is one of the main clinical features of MetS in post-menopausal patients with T2DM . Weight In the current study, post-menopausal women had higher systolic blood pressure compared to pre-menopausal women with T2DM (P <0.005) but there was no significant difference in diastolic blood pressure between the groups. Franklin et al.  showed tA twofold increase in CVD risk has been reported in post-menopausal women compared to pre-menopausal women . HoweverFurthermore, diabetes can attenuate protective effects of oestrogen in premenopausal status . StudiesThe main limitation of the present study was the cross-sectional study design used, which precludes the determination of the direction of causality. However, this is the first study to compare MetS in pre- and post-menopausal diabetic women.In Conclusion, MetS must be evaluated in diabetic patients regardless of the time of menopausal transition . In pati